To date, there has been no information about the long-term replication mechanisms of the beta papillomaviruses. We have found that the beta HPV E2 proteins (HPV 5 and 8) target the peri-centromeric region of mitotic chromosomes. We have shown that these speckles do not contain Brd4 and, unlike BPV1, the N-terminal Brd4-interacting domain of HPV8 E2 is not required for chromosome binding. The HPV8 E2 protein targets the short arm of human acrocentric mitotic chromosomes, a region between the centromere and telomere. The E2 protein interacts with the repeated ribosomal DNA genes found in this location and co-localizes with UBF, the RNA polymerase I transcription factor. Therefore, HPV8 E2 genome tethering occurs by a Brd4-independent mechanism through a novel interaction with specific regions of mitotic chromosomes. We have found that the domains of HPV8 E2 required for chromosomal association are also quite different from BPV1 E2, further supporting the finding that these proteins have different targets. The hinge and the DNA binding domain of HPV8 E2 are required for this association. The HPV8 hinge is very long, compared to other PV E2s, and has an unusual sequence composition rich in arginine, glycine, serine and proline. We have mapped a short 16 amino acid peptide from the hinge region that is necessary and for interaction with mitotic chromosomes. This 16 amino acid region contains an RXXS motif that is highly conserved among beta-papillomaviruses and both the arginine and serine residues within this motif are required for mitotic chromosome binding. We have also shown that a similar region from the beta HPV5 E2 protein also mediates chromosomal binding. We also find that the HPV8 E2 proteins are highly phosphorylated and the RXXS motif is a site of phosphorylation. The HPV8 E2 chromosome binding sequence also has sequence similarity with chromosome binding regions in the gamma herpesvirus EBNA and LANA tethering proteins.